多房棘球绦虫ELP重组蛋白疫苗免疫Balb/c小鼠引起的免疫应答

Immune response in mice induced by recombinant protein ELP vaccine of Echinococcus multilocularis

目的 观察多房棘球绦虫ELP重组蛋白疫苗免疫Balb c小鼠引起的体液和细胞免疫应答。方法 在成功构建多房棘球绦虫elp基因原核表达载体 (pQ ELP)的基础上 ,以亲和层析法纯化重组蛋白 ,SDS PAGE鉴定 ,Bradford法进行定量。用ELP重组蛋白 (A组 )及ELP重组蛋白加弗氏佐剂 (B组 )免疫Balb c小鼠 (10只 组 ) ,以生理盐水 (NS组 )作对照。ELISA方法检测免疫后不同时间产生的特异性IgG1,IgG2a和IgG2b水平。双抗体夹心ELISA试剂盒检测免疫鼠脾脏单个核细胞 (PMNC)在受到特异抗原刺激后 ,产生IL 4、IL 12和IFN γ的能力 ,3H TdR掺入法检测脾淋巴细胞的增殖能力。结果 本研究成功获得高纯度重组蛋白ELP。首次免疫后 2周 ,A、B组小鼠均产生了大量的特异IgG1抗体 ,B组还产生了少量的特异IgG2b抗体。除B组小鼠脾PMNC产生了微量IFN γ外 ,A组、NS组IFN γ及各组IL 4和IL 12均未检出。A、B组小鼠脾淋巴细胞增殖能力增高 ,其淋巴细胞CPM值分别为NS组的 2 .8和 10 .8倍 ,受到多房棘球绦虫抗原或刀豆素刺激时 ,A、B组淋巴细胞增殖更明显。结论 多房棘球绦虫ELP重组蛋白疫苗可引起很强的体液免疫应答和微弱的细胞免疫应答 。

Objective To observe the humoral and cellular immune responses in Balb/c mice induced by the recombinant protein ELP vaccine of Echinococcus multilocularis.Methods Based on the successful construction of pQ-ELP, the prokaryotic expression vector of E. multilocularis elp gene, the author produced and purified the recombinant protein ELP expressed in E. coli SG13009. Quality of purification and concentration of ELP were determined with SDS-PAGE and Bradford method respectively. Three groups of 10 Balb/c mice, namely Group A, B and NS, were immunized with ELP or ELP and Freunds adjuvant or normal saline as control. At different times after immunization, specific IgG1, IgG2a and IgG2b were detected with ELISA method. The IFN-γ, IL-4 and IL-12 produced by peripheral mononuclear cell(PMNC) of spleen after stimulation of E. multilocularis antigen were examined with Double-Antibody-Sandwich ELISA kits. The effects of ELP vaccine on the proliferation of spleen lymphocytes were evaluated by 3 H-TdR methods.Results Highly pure recombinant protein ELP was obtained. Two weeks after the first dose immunization, a high level of specific IgG1 was detected in both Group A and Group B, a low level of IgG2b was detectable in Group B as well. Except for small quantity of IFN-γ detected in Group B, no cytokine was detected in all other cytokine assays. Compared with that of Group NS, the proliferation of spleen lymphocytes of both Group A and B were enhanced. Their CPM values were 2.8 and 10.8 times as high as that of Group NS. The capacity of lymphocyte proliferation was further increased after in vitro stimulation of E. mulitilocularis antigen or ConA.Conclusion The recombinant protein ELP vaccine of E. multilocularis is able to induce strong humoral immune response and weak cellular immune response in Balb/c mice, and cellular immune response can be further enhanced if the vaccine is combined with Freuns adjuvant.